The present invention relates to a chain-shortened polynucleotide particularly useful as a medicine, and a method for preparing the same. More specifically, the present invention relates to a synthetic chain-shortened polynucleotide or salts thereof, wherein the proportion of a 2xe2x80x2-5xe2x80x2 phosphodiester bond is up to 3% based on the whole phosphodiester bonds, i.e., the rate of phosphate groups rearranged from 3xe2x80x2 position to 2xe2x80x2 position based on the whole phosphate groups of phosphodiester bonds (the phosphate rearrangement rate) is up to 3%, and a method for preparing the same.
Polynucleotides typified by polyinosinicxe2x80x94polycytidylic acid, i.e., poly(I)xc2x7poly(C), are well-known compounds in the art, and the potentiality as a medicine for treating hepatitis or cancer have been investigated in view of their interferon inducing action, immune activating action, and the like.
The pharmacological action of these polynucleotides has high correlation with the chain length, and longer the chain length, stronger the interferon inducing action and the like. On the other hand, longer the chain length, stronger the toxicity manifested.
Recently, there has been an approach for reducing toxicity with maintaining useful pharmacological action of a polynucleotide, by a method wherein a synthetic polynucleotide having a relatively shorter chain prepared by the hydrolysis of a polynucleotide is enclosed in a carrier such as cationic liposome effective for introducing a medicament into a cell (e.g., PCT W099/20283, PCT WO99/4853 1).
It is known that when a polynucleotide is hydrolyzed to shorten the chain length as described above, some phosphate groups cause intramolecular rearrangement from 3xe2x80x2 position to 2xe2x80x2 position through a mechanism called pseudo rotation simultaneously with the chain-shortening (see, e.g., xe2x80x9cProtein Nucleic acid Enzymexe2x80x9d, Vol. 40, No. 10, pp. 1323 to 1332 (1995)). As a result, a portion of 3xe2x80x2-5xe2x80x2 phosphodiester bonds in the chain-shortened polynucleotide molecule are replaced by 2xe2x80x2-5xe2x80x2 phosphodiester bonds. It has never been known whether or not such a phosphate rearrangement phenomenon would affect the pharmacological action.
An object of the present invention is to provide, firstly, a chain-shortened polynucleotide or salt thereof and a double stranded chain-shortened polynucleotide or salt thereof, which are safe and effective as a medicine.
The present inventors have been intensively studied and found that the problems described above can be solved by a chain-shortened polynucleotide which contains 2xe2x80x2-5xe2x80x2 phosphodiester bond produced mainly in a chain-shortening reaction only at a particular proportion or less, or a salt thereof, and accomplished the present invention.
An aspect of the present invention is a chain-shortened polynucleotide containing a 2xe2x80x2-5xe2x80x2 phosphodiester bond at a proportion of 3% or less, preferably 2% or less, based on the whole phosphodiester bonds, or salt thereof.
The present invention includes also as an embodiment a double stranded chain-shortened polynucleotide or salt thereof, which is formed from two chain-shortened polynucleotides or salts thereof capable of forming a double strand, which is inclusive in the above-described chain-shortened polynucleotides containing a 2xe2x80x2-5xe2x80x2 phosphodiester bond at a proportion of 3% or less, preferably 2% or less, based on the whole phosphodiester bonds. Further, the present invention also includes a composition comprising a complex formed from a carrier effective for introducing a medicament into a cell and the above-described chain-shortened polynucleotide or salt thereof wherein the proportion of a 2xe2x80x2-5xe2x80x2 phosphodiester bond is 3% or less based on the whole phosphodiester bonds, or the double stranded chain-shortened polynucleotide or salt thereof formed from two chain-shortened polynucleotides or salts thereof capable of forming a double strand as an essential ingredient.
The polynucleotide used in the present invention is a compound comprising at least about 20 nucleotides, which is formed by polymerizing linearly through a phosphodiester bond and includes synthetic and natural compounds. Specific examples include polyinosinic acid [namely, poly(I)] or an analogue thereof, polycytidylic acid [namely, poly(C)] or an analogue thereof, polyadenylic acid [namely, poly(A)] or an analogue thereof, and polyuridylic acid [namely, poly(U)] or an analogue thereof.
The polyinosinic acid analogue is a homopolymer in which all or a part of inosinic acid is chemically modified or a copolymer of inosinic acid with other nucleotide, for example, poly(7-deazainosinic acid) and poly(2xe2x80x2-azidoinosinic acid). The polycytidylic acid analogue is a homopolymer in which all or a part of cytidylic acid is chemically modified or a copolymer of cytidylic acid with other nucleotide, for example, poly(5-bromocytidylic acid), poly(2-thiocytidylic acid), poly(cytidine-5xe2x80x2-thiophosphoric acid), poly(cytidylic acid, uridylic acid), poly(cytidylic acid, 4-thiouridylic acid) and poly(1-vinylcytidylic acid). The polyadenylic acid analogue and polyuridylic acid analogue are defined likewise. Among them, polyinosinic acid and polycytidylic acid are suitable in the present invention.
The average chain length of the chain-shortened polynucleotide of the present invention is suitably from 0.1 k bases to 1 k base. The term xe2x80x9cbasexe2x80x9d means the number of base and xe2x80x9c1 k basexe2x80x9d indicates a base number of 1000, and hereinafter, xe2x80x9cbase(s)xe2x80x9d is abbreviated to xe2x80x9cbxe2x80x9d. The mean chain length is preferably from 200 b to 800 b, and more preferably from 300 b to 600 b. The average chain length can be determined easily, for example, by gel permeation chromatography (hereinafter, referred to as xe2x80x9cGPCxe2x80x9d) as described hereinafter in Experiment 5.
In the chain-shortened polynucleotide of the present invention, the phosphate rearrangement rate is 3% or less, preferably 2% or less or between 0.1% and 2%, and more preferably 1% or less or between 0.1% and 1%.
The rearrangement of phosphate group from 3xe2x80x2 position to 2xe2x80x2 position in polynucleotide can be confirmed easily, for example, by a method as described in Experiment 6. Namely, a polynucleotide is degraded with nuclease P1, which specifically hydrolyzes a 3xe2x80x2-5xe2x80x2 phosphodiester bond to such levels as a nucleoside, nucleotide and oligonucleotide, and then treated with an alkaline phosphatase, which specifically hydrolyzes a terminal phosphate group, to convert the whole nucleotides into nucleosides. On the other hand, an oligonucleotide having a 2xe2x80x2-5xe2x80x2 phosphodiester bond, which is not hydrolyzed by the nuclease P1 is not degraded till nucleoside even by the treatment with an alkaline phosphatase because an intramolecular 2xe2x80x2-5xe2x80x2 phosphodiester bond is not hydrolyzed. The phosphate rearrangement rate can be calculated by determining nucleosides and oligonucleotides (most of them are dimers) by liquid chromatography, or the like.
Examples of a pair of chain-shortened polynucleotides capable of forming a double strand in connection with the present invention include polyinosinic acid and polycytidylic acid, polyadenylic acid and polyuridylic acid, polyinosinic acid analogue and polycytidylic acid, polyinosinic acid and polycytidylic acid analogue, polyinosinic acid analogue and polycytidylic acid analogue, polyadenylic acid analogue and polyuridylic acid, polyadenylic acid and polyuridylic acid analogue, and polyadenylic acid analogue and polyuridylic acid analogue. Therefore, examples of the double stranded chain-shortened polynucleotide formed between two chain-shortened polynucleotides capable of forming a double strand include polyinosinic-polycytidylic acid, polyadenylic-polyuridylic acid, polyinosinic analogue-polycytidylic acid, polyinosinic-polycytidylic acid analogue, polyinosinic analogue-polycytidylic acid analogue, polyadenylic analogue-polyuridylic acid, polyadenylic-polyuridylic acid analogue, and polyadenylic analogue-polyuridylic acid analogue. For the purposes of the present invention, polyinosinic-polycytidylic acid would be a preferred double stranded chain-shortened polynucleotide.
The average chain length of the above-described double stranded chain-shortened polynucleotide is reasonably regarded as corresponding to the average chain length of the whole chain-shortened polynucleotides. Accordingly, the latter can be used to show the apparent average chain length of the double stranded chain-shortened polynucleotide in terms of the number of base pairs (bp). Therefore, the average chain length of the above-described double stranded chain-shortened polynucleotide is from 0.1 k bp to 1 k bp. The term xe2x80x9cbpxe2x80x9d means the number of base pairs and xe2x80x9c1 k bpxe2x80x9d corresponds to a base pair number of 1000. The average chain, length of the double-stranded polynucleotide is preferably from 200 bp to 800 bp, and more preferably from 300 bp to 600 bp.
The salt of a chain-shortened polynucleotide and that of a double stranded chain-shortened polynucleotide of the present invention are not particularly restricted as far as they are pharmaceutically acceptable, and examples thereof include a sodium salt and potassium salt.
As a carrier effective for introducing a medicament into a cell, those having a positive charge are exemplified, and specific examples include cationic polymers such as poly-L-lysine, cationic liposomes such as Lipofectin(copyright), Lipofectamine(copyright), Lipofectace(copyright), DMRIE-C(copyright), etc., and carriers considered to be of the same kind which are disclosed in PCT WO94/19314. The carriers for carrying a medicament are formed, for example, from 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoyl glycerol of the formula [I]: 
and a phospholipid (e.g., phosphatidyl choline, phosphatidyl ethanolamine, yolk lecithin, soy bean lecithin, hydrogenated phospholipid thereof) as essential structural components.
It is believed that the above-described cationic liposome is positively charged and forms an electrostatic complex with a negatively charged polynucleotide or oligonucleotide. When the resultant complex fuses with cell membrane, the polynucleotide or oligonucleotide is concurrently introduced into the cell. Such a complex is sometimes called xe2x80x9clipoplexxe2x80x9d.
A method for preparing the chain-shortened polynucleotide of the present invention will be described in detail. The chain-shortened polynucleotide of the present invention can be prepared, for example, by hydrolyzing the starting polynucleotide in solution at suitable pH range while heating at a suitable temperature range. The suitable pH of the aqueous solution in this procedure is basic, i.e., pH 7 or more, preferably pH 7 to 10. Considering the reaction rate of the chain-shortening reaction and the stability of base moiety, more preferable pH of the solution would be between 8 and 9. The reaction temperature is suitably within a range of 20 to 110xc2x0 C., preferably 40 to 100xc2x0 C., from the standpoint of the stability of a base. However, considering the sufficient hydrolysis rate and the stability of base moiety, more preferable reaction temperature would be between 50 and 90xc2x0 C.
More specifically, for example, a polynucleotide is dissolved in water such as injectable water, distilled water for injection or physiological saline with stirring, and pH of the solution is adjusted to 8 to 9 with a buffer or a pH regulator. When the hydrolysis is conducted by heating the solution at reaction temperature ranging from 50 to 90xc2x0 C. for 0.5 to 60 hours while monitoring the average chain length and the phosphate rearrangement rate, a chain-shortened polynucleotide containing less phosphate groups rearranged and having an average chain length between 0.1 kb and 1 kb can be produced.
Pharmaceutically acceptable additives such as a buffer or a pH regulator may be used for adjusting pH. Specific examples include buffers and pH regulators such as aminoacetic acid (synonym: glycine), tris(hydroxymethyl)aminomethane (synonym: Tris), sodium carbonate, sodium hydrogen carbonate (synonym: sodium bicarbonate), sodium hydroxide, diethanolamine, triethanolamine and the like. There are no limitations regarding the kind, combination, concentration and the like of these additives.
Monomers and unnecessary salts, impurities, by-products produced during the chain-shortening reaction and the like can be removed from the system by treating the reaction solution by dialysis or with activated carbon.
The chain-shortened polynucleotide of the present invention can also be prepared by treating the starting polynucleotide in solution with a phosphodiesterase at suitable pH range while heating at a suitable temperature range. The suitable pH of the aqueous solution in this procedure is between 4 and 9, preferably, between 5 and 8. Considering the phosphate rearrangement during the chain-shortening reaction, more preferable pH of the solution would be between 6 and 7. The reaction temperature is suitably within a range of 20 to 60xc2x0 C., preferably 25 to 50xc2x0 C., from the standpoint of the characteristics of the enzyme. However, more preferable reaction temperature would be between 30 and 40xc2x0 C. taking the sufficient hydrolysis rate, the avoidance of influence of non-enzymatic hydrolysis such as hydrolysis with heat, and the prevention of phosphate-group rearrangement into consideration.
More specifically, for example, a polynucleotide is dissolved in water such as injectable water, distilled water for injection or physiological saline with stirring. The pH of the solution is optionally adjusted by adding a buffer or a pH regulator, if necessary. To the solution is added phosphodiesterase such as nuclease P1 and the resultant mixture is subjected to the chain-shortening reaction at temperature from 30 to 40xc2x0 C. while monitoring the average chain length and phosphate rearrangement rate to obtain a chain-shortened polynucleotide containing less phosphate groups rearranged and having an average chain length between 0. 1 kb and 1 kb. There are no limitations regarding the enzyme concentration or reaction conditions.
Monomers and unnecessary salts, impurities, by-products produced during the chain-shortening reaction and the like can be removed from the system by treating the reaction solution by the ethanol precipitation method, dialysis or with activated carbon.
The chain-shortened polynucleotide can be purified by an appropriate separation method with a membrane. For example, membrane ultrafiltration is suited for the purpose of fractionating polynucleotides of average chain length between 0.1 kb and 1 kb of the present invention. There are no limitations regarding the quality of material or the pore size of the membrane.
As a starting material, any polynucleotides can be used regardless of origin such as natural or synthetic, the kind of salt or the chain length. Examples of natural polynucleotide include tRNA and polyadenylic acid. On the other hand, a synthetic polynucleotide can be produced from an RNA synthetase such as polynucleotide phosphorylase or immobilized enzymes thereof. Further, sodium polyinosinate, sodium polycytidylate, etc., which are commercially available as interferon inducing reagents, are also usable as a starting material.
The double stranded chain-shortened polynucleotide of the resent invention can be prepared by mixing, in a suitable solution (for example, 0.15 M NaCl-containing 10 mM Tris-hydrochloric acid buffer (Tris-HCl buffer, pH7)), two chain-shortened polynucleotides capable of forming a double strand among chain-shortened polynucleotides containing less phosphate groups rearranged as prepared in the above, or alternatively, by allowing them to anneal in a conventional manner. As the annealing method, there is, for example, a method in which a solution containing two chain-shortened polynucleotides capable of forming a double strand is heated up to 70 to 80xc2x0 C., and then cooled gradually.
A chain-shortened polynucleotide with less phosphate groups rearranged or a double stranded chain-shortened polynucleotide with less phosphate groups rearranged obtained as described above can be treated by lyophilization to give a lyophilized product storable for a long period. The lyophilization treatment can be conducted in a conventional manner. For example, a lyophilized product can be obtained as follows: a solution of a chain-shortened polynucleotide obtained under the conditions above is sterilized by filtration, the filtrate is then poured on a metal bat previously treated by dry heat sterilization, a pre-freezing is conducted at a shelf temperature from xe2x88x9240 to xe2x88x9220xc2x0 C. for about 1 to 4 hours, and primary drying is conducted before the secondary drying which is effected under reduced pressure at a shelf temperature from 15 to 30xc2x0 C. (for about 10 to 50 hours). Generally, such a lyophilized product can be used after reconstitution (re-dissolution) by the addition of an appropriate solution such as injectable water, distilled water for injection, physiological saline, maltose solution, glucose solution, or the like.
The composition of the present invention can be prepared in a manner similar to those generally used for the preparation of liposome, which composition comprises a complex (hereinafter, referred to as the present complex) formed with a carrier effective for introducing a medicament into a cell and a two chain-shortened polynucleotide containing 2xe2x80x2-5xe2x80x2 phosphodiester bonds at proportion of 3% or less based on the whole phosphodiester bonds and being capable of forming a double strand, or a double-stranded chain-shortened polynucleotide formed between the said two chain-shortened polynucleotides capable of forming a double strand as an essential ingredient. Specifically, a composition for injection of the present invention can be prepared by the following steps comprising adding water (injectable water, distilled water for injection, physiological saline and the like) to a carrier effective for introducing a medicament into a cell, for example, a cationic liposome or raw material thereof (e.g., glycerol derivative such as 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoyl glycerol and the like and phospholipid); stirring the mixture; treating the mixture with a suitable device for dispersion, for example, a homomixer, a homogenizer, a ultrasonic dispersing device, a ultrasonic homogenizer, a high pressure emulsifying dispersing device, Microfluidizer (trade name), Nanomizer (trade name), De Bee 2000 (trade name), Ultimizer (trade name) or Manton-Gaulin type high pressure homogenizer; adding a chain-shortened polynucleotide or a double stranded chain-shortened polynucleotide of the present invention to the resultant lipid dispersion; re-dispersing the mixture by treating with a suitable dispersing device; and then subjecting the resultant composition to sterilization by filtration or the like. Any other additives can be added at an appropriate step during the preparation without any particular limitation. Alternatively, a composition of the present invention containing the present complex can be prepared as follows. Thus, a mixture of a carrier effective for introducing a medicament into a cell, for example, a cationic liposome or raw material thereof (e.g., glycerol derivative such as 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoyl glycerol and the like and phospholipid) and a chain-shortened polynucleotide or a double stranded chain-shortened polynucleotide of the present invention is first prepared. To the previously prepared mixture is added water while conducting the dispersion treatment at the same time. Further, a composition of the present invention can also be prepared via a suitable crude dispersing step during the above-described methods.
The resultant composition of the present invention can be lyophilized, whereby a lyophilized preparation of a composition of the present invention storable for a long period is obtained. The lyophilization can be conducted in a conventional manner. For example, a given amount of a composition of the present invention, which has been obtained by the above-mentioned dispersion and sterilization by filtration, is dispensed into a vial. The lyophilization is carried out by subjecting the vial to pre-freezing at a temperature between about xe2x88x9240 and xe2x88x9220xc2x0 C. for about 2 to 3 hours, the primary drying at a temperature between about 0 and 10xc2x0 C. under reduced pressure, and the secondary drying at a temperature between about 15 and 25xc2x0 C. under reduced pressure. Generally, after replacing the inner space with an inert gas such as nitrogen gas, the vial is capped to provide a lyophilized preparation of a composition of the present invention.
The lyophilized preparation of a composition of the present invention can be used after reconstitution by adding an appropriate solution before use. Examples of such a solution for reconstitution include injectable water, distilled water for injection, physiological saline, maltose solution, glucose solution and other general infusion solutions and the like.
The composition of the present invention and lyophilized preparation thereof can be used in the form of a pharmaceutical preparation as a medicine. The composition of the present invention and lyophilized preparation as a medicine exhibits a pharmacological activity owing to the active polynucleotide. The specific examples of the medicine include interferon inducing agents, immune activating agents, intracellular nuclease activating agents, cancer treating or preventive agents, and hepatitis treating or preventive agents.